Neuronal survival in developing animals is often dependent on afferent activity. In the posthatch chick, elimination of eighth nerve activity leads to the death of 20-40% of the neurons in the cochlear nucleus, nucleus magnocellularis (NM). The factors that determine whether an individual NM neuron will live or die are largely unknown. Previous studies indicate that both cell death and cell survival mechanisms compete to determine an individual cell's fate. This proposal seeks to examine the underlying cellular and molecular cascades that are activated following deafferentation. Several studies have implicated bcl-2 family members in the regulation of common cell death pathways. This gene family includes both cell "protectors" (bcl-2 and bcl-xL) and "executioners" (bax and bak). The effect of deafferentation on the activation and expression of pro-survival bcl-2 gene and gene product will be examined using both in situ hybridization and immunocytochemistry. Additional studies will be performed to assess the effect of deafferentation on other members of the bcl-2 family. For example, pro-apoptotic bax will be investigated for differential expression from the pro-survival bcl-2 protein. Another line of research will focus on molecules thought to participate in apoptosis downstream of bcl-2 genes. Mitochondrial integrity is pivotal to the maintenance of healthy cells; compromise of mitochondrial bioenergetics can result in the release of cytochrome-c. Cytosolic cytochrome-c activates what is commonly referred to as the apoptosome. This complex of molecules including cytochrome-c, Apaf-1, and caspase-9 will initiate proteolytic caspase cascades that ultimately lead to cell death. Afferent regulation of all the molecules involved in the apoptosome will be investigated through a combination of immuncytochemistry and Western blotting techniques. It is the hope that this research will elucidate some of the underlying mechanisms involved with deafferentation-induced cell death.